Many areas of modem biomedical research focus on signaling networks that mediate cellular responses to stimuli. The chemotaxis system of Escherichia coli provides an excellent model system for exploring how sophisticated sensory-response systems work at a molecular level. CheA, an autophosphorylating protein kinase, plays a central role in the chemotaxis signal transduction pathway: it regulates (via phosphorylation) two downstream effector proteins, CheY and CheB. P-CheY, in turn, determines the pattern of flagellar rotation. The catalytic activity of CheA is regulated by cell-surface receptor proteins that respond to altered concentrations of attractant and repellent chemicals in the extracellular environment. As a result of this regulation, the level of phosphorylated CheY is modulated in response to chemotactic stimuli on a rapid (sub-second) time scale. The long-term goal of this project is to define how this modulation results from interactions among the Che proteins and receptors. Experiments in this application will focus on three key aspects of the signaling biochemistry: (1) Signaling dynamics--A reconstituted system utilizing vesicleassociated signaling complexes (receptor-CheW-CheA) will be used to examine the magnitude and kinetics of responses to rapid, stepwise increases of the attractant aspartate. Photorelease of caged aspartate will be used to generate aspartate concentration jumps, and rapid fluorescence measurements (in a flash-photoloysis instrument) will monitor ensuing changes in CheY phosphorylation. (2) Effector recognition by CheA-Mutant CheA proteins with alterations in the CheY/CheB binding domain will be identified by two different approaches. The effects of these mutations on the affinity and kinetics of CheA interactions with CheY and CheB will be defined using in vitro binding titrations and rapid-reaction methods (stopped-flow and rapid-quench). (3) Mechanism of receptor-mediated regulation of CheA -Two aspects of receptor-mediated regulation of CheA will be explored. First, a fluorescence method (FRET) will be adapted to monitor CheA conformational changes that shift the relative position of different functional domains in CheA. This approach will provide a powerful way of investigating whether specific conformational changes underly regulation of CheA autokinase activity. Second, the role of CheW in mediating this regulation will be explored by studying mutant CheW proteins that have altered affinity for CheA or receptor. The abilities of these mutant CheW proteins to mediate regulation of CheA activity in vitro and to support chemotaxis in vivo will be determined.